In the DNA and genetic engineering arts, there is a critical need for novel site-specific DNA cleavage agents that: (1) cleave DNA at long DNA recognition sites, and (2) have little or no non-specific DNA cleavage activity (i.e., have little or no DNA cleavage activity at DNA sites other than the DNA recognition sites). The invention disclosed herein meets this need.
Prior to 1970, there was no known technology for cleaving a double-stranded DNA molecule into discrete defined fragments. However, in 1970, a class of naturally occurring enzymes, termed "type II restriction endonucleases", was discovered. Type II endonucleases bind to specific DNA recognition sites in a double-stranded DNA molecule and cleave the DNA within, or proximate to, the specific DNA recognition sites to give rise to discrete DNA fragments of defined length and sequence. Type II restriction endonucleases are fundamentally important in the manipulation of DNA; present day DNA and genetic engineering technologies are dependent upon these restriction endonucleases. Hundreds of type II restriction endonucleases have now been isolated from a wide variety of bacteria and other microorganisms. (Nucl. Acids Res. 16:R271-R313, 1988.) All known type II restriction endonucleases have DNA recognition sites less than or equal to 8 base pairs in length. Most type II restriction endonucleases have DNA recognition sites of 4 or 6 base pairs in length. A very few known type II restriction endonucleases have DNA recognition sites of 8 base pairs in length.
The fact that the known type II endonucleases have short DNA recognition sites is an important limitation. The length of the specific DNA recognition site of the site-specific cleavage agent determines the frequency of occurrence of the DNA recognition site in random-sequence DNA (and thus the average fragment length of DNA that is produced upon cleavage). The frequency of occurrence of a DNA recognition site in random-sequence DNA is 2/4.sup.N, where N is the DNA recognition site length in base pairs (for an asymmetric DNA recognition site; 1/4.sup.N for a 2-fold symmetric DNA recognition site; see Proc. Natl. Acad. Sci. USA 83:1608-1612, 1986). Consequently, a particular 4 base pair DNA recognition site should occur once in every 4.sup.4 /2, (i.e., 128) base pairs in a random DNA sequence. A particular 6 base pair DNA recognition site should occur once in every 4.sup.6 /2, (i.e., 2,048) base pairs in a random DNA sequence. A particular 8 base pair DNA recognition site should occur once in every 4.sup.8 /2, (i.e., 32,768) base pairs in a random DNA sequence. With small pieces of DNA, the known natural type II restriction endonucleases give rise to a small number of DNA fragments. However, with large pieces of DNA, such as human chromosomes, the known type II restriction endonucleases give rise to an unmanageably large number of DNA fragments.
In order efficiently to manipulate and map large pieces of DNA, such as human chromosomes, it would be useful to have site-specific DNA cleavage agents that have DNA recognition sites longer than those of the known type II restriction endonucleases (i.e., DNA recognition sites greater than or equal to nine base pairs in length). Therefore, several efforts have been made to construct synthetic or semi-synthetic site-specific DNA cleavage agents that have DNA recognition sites greater than or equal to nine base pairs in length.
In prior art, several synthetic or semi-synthetic site-specific DNA cleavage agents, that have DNA recognition sites greater than or equal to nine base pairs in length have been constructed by covalently attaching a nucleolytic moiety to a sequence-specific DNA binding molecule having a DNA recognition site greater than or equal to nine base pairs in length (Science 237:1197, 1987; J. Amer. Chem. Soc. 112:4579, 1990; J. Amer. Chem. Soc. 113:5446, 1991; Proc. Natl. Acad. Sci. USA 87:2882, 1990; Science 238:645, 1987; Proc. Natl. Acad. Sci. USA 86:9702, 1989; J. Amer. Chem. Soc. 110:7927, 1988; Science 249:73, 1990; Proc. Natl. Acad. Sci. USA 87:9858, 1990). The following approaches have been employed: incorporation of a nucleolytic moiety at multiple, random amino acids within the sequence-specific DNA binding protein Trp repressor (Science 237:1197, 1987); incorporation of a nucleolytic moiety at amino acid 32 of the sequence-specific DNA binding protein lambda repressor(1-102) (J. Amer. Chem. Soc. 112:4579, 1990); incorporation of a nucleolytic moiety at amino acid 66 of the sequence-specific DNA binding protein lambda cro (J. Amer. Chem. Soc. 113:5446, 1991); incorporation of a nucleolytic moiety at amino acid 178 of sequence-specific DNA binding protein CAP (Proc. Natl. Acad. Sci. USA 87:2882, 1990); and incorporation of a nucleolytic moiety in an oligonucleotide able to form triple helix (Science 238:645, 1987; Proc. Natl. Acad. Sci. USA 86:9702, 1989; J. Amer. Chem. Soc. 110:7927, 1988; Science 249:73, 1990; Proc. Natl. Acad. Sci. USA 87:9858, 1990).
In most examples in prior art, the nucleolytic moiety utilized was a chelator or a chelator-metal complex (Science 237:1197, 1987; J. Amer. Chem. Soc. 113:5446, 1991; Proc. Natl. Acad. Sci. USA 87:2882, 1990; Science 238:645, 1987; Proc. Natl. Acad. Sci. USA 86:9702, 1989; J. Amer. Chem. Soc. 110:7927, 1988; Science 249:73, 1990). Certain chelator-metal complexes, for example, 1,10-phenanthroline:Cu and ethylenediaminetetraacetate:Fe, are able to cleave DNA in an essentially random, sequence-independent fashion (Acc. Chem. Res. 19:180, 1986; J. Amer. Chem. Soc. 104:31 3, 1982; Science 230:679, 1985). It is possible to target the DNA cleavage activity of chelator-metal complexes to specific DNA sites by covalently attaching the chelator-metal complex to a sequence-specific DNA binding molecule (J. Amer. Chem. Soc. 104:6861, 1982).
However, prior-art synthetic or semi-synthetic site-specific DNA cleavage agents have exhibited significant non-specific DNA cleavage activity (i.e., they have exhibited significant DNA cleavage activity at DNA sites other than the specific DNA recognition sites). Non-specific DNA cleavage activity has precluded the practical use of prior-art synthetic or semi-synthetic site-specific DNA cleavage agents. In particular, non-specific DNA cleavage activity has precluded the practical use of prior-art synthetic or semi-synthetic site-specific DNA cleavage agents for cleavage of large pieces of DNA, such as human chromosomes, since with large pieces of DNA the ratio of non-specific DNA sites to specific DNA recognition sites is high. (Non-specific DNA sites are defined as DNA sites other than the specific DNA recognition sites. The number of non-specific DNA sites in a piece of DNA is equal to L - N, where L is the length of the piece of DNA in base pairs, and N is the length of the DNA recognition site in base pairs [Proc. Natl. Acad. Sci. USA 83:1608, 1986].)
All sequence-specific DNA binding molecules bind with detectable affinity to non-specific DNA sites (Proc. Natl. Acad. Sci. USA 83:1608, 1986). Prior-art synthetic or semi-synthetic site-specific DNA cleavage agents have exhibited significant non-specific DNA cleavage activity because they have been able to cleave DNA when bound at non-specific DNA sites.
The invention disclosed herein enables production of novel site-specific DNA cleavage agents that have DNA recognition sites greater than or equal to nine base pairs, and that have little or no ability to cleave DNA when bound at non-specific DNA sites.